Methods for analysis of protamine

ABSTRACT

The invention provides a method for analyzing a protamine sample for the presence or amount of at least one nucleotidic impurity. The invention also provides a method for the quantitation of peptides in a sample of protamine comprising four major peptides and at least one related impurity.

BACKGROUND OF THE INVENTION

Protamines are simple proteins of low molecular weight that are rich inarginine and strongly basic. Protamines are extracted from the sperm ofsalmon and other fish species.

Protamine sulfate occurs as a fine white, or off white, amorphous orcrystalline powder. It is sparingly soluble in water. The pH ofprotamine sulfate in solution is between 6.0 and 7.0. It is used to bindwith heparin in the body. The cationic protamine at a pH of 6.8 to 7.1reacts with anionic heparin at a pH of 5.0 to 7.5 to form an inactivecomplex.

Protamine sulfate may be supplied as a sterile, isotonic solution foradministration to patients. It acts as a heparin antagonist and is alsoa weak anticoagulant.

When supplied as a pharmaceutical active ingredient, protamine sulfatecontains four major peptides and other related peptides (or possibleimpurities). Presently, there are no reference standards available(including impurity standards). In addition, protamine sulfate maycontain impurities in the form of nucleotides that in turn may derivefrom DNA present in the sample.

It is necessary to characterize protamine sulfate in order to satisfyvarious regulatory requirements before use in humans and other animals.Presently, there are no reference standards of the four major peptidescomprising protamine sulfate or of related peptides or possibleimpurities. Thus, there remains in the art a need for improved methodsfor the analytical characterization of protamine sulfate.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method for analyzing a protamine sample for thepresence or amount of at least one nucleotidic impurity, comprising thesteps of (a) providing a protamine sample, (b) subjecting the sample ofprotamine to disruption with a protamine-disrupting agent to provide afirst mixture, (c) subjecting the first mixture to a digestion step toprovide a second mixture, and (d) determining the presence or amount ofthe at least one nucleotidic impurity present in the second mixture.

The invention also provides a method for analyzing a sample of protaminecomprising four major peptides corresponding to Peptides 1-4 in FIG. 1and at least one related impurity, comprising (a) subjecting a solutionof the sample of protamine to a high pressure liquid chromatography(HPLC) under conditions suitable to resolve the four major peptides andat least one related impurity in a chromatogram, (b) determining theareas under the peaks of the four major peptides in the chromatogram,(c) determining the area under the peak of the at least one relatedimpurity in the chromatogram, and (d) determining the concentration of(i) one or more of the four major peptides, (ii) the sum of two or moreof the four major peptides, and/or (iii) the at least one relatedimpurity.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 depicts a high pressure liquid chromatogram of a sample ofprotamine sulfate analyzed in accordance with an embodiment of theinvention.

FIG. 2 depicts representative chromatograms of Nucleoside IdentificationSolution showing nucleotidic impurities of DNA and RNA (FIG. 2A);Protamine Sulfate, USP Raw Material from Source 1 (FIG. 2B); ProtamineSulfate, USP Raw Material from Source 2 (FIG. 2C); and ProtamineSulfate, USP Raw Material from Source 3 (FIG. 2D).

FIG. 3 depicts an expanded scale high pressure liquid chromatogram ofthe chromatogram depicted in FIG. 1.

FIG. 4 depicts a high pressure liquid chromatogram of a sample ofprotamine sulfate analyzed by a comparative method.

FIG. 5 depicts an expanded scale high pressure liquid chromatogram ofthe chromatogram depicted in FIG. 4.

FIG. 6 depicts high pressure liquid chromatograms of a sample ofprotamine sulfate analyzed in accordance with an embodiment of theinvention (FIG. 6A) and a comparative method (FIG. 6B).

DETAILED DESCRIPTION OF THE INVENTION

In an embodiment, the invention provides a method for analyzing aprotamine sample for the presence or amount of at least one nucleotidicimpurity, comprising the steps of (a) providing a protamine sample, (b)subjecting the sample of protamine to disruption with aprotamine-disrupting agent to provide a first mixture, (c) subjectingthe first mixture to a digestion step to provide a second mixture, and(d) determining the presence or amount of the at least one nucleotidicimpurity present in the second mixture.

In any of the embodiments of the methods described herein, the protaminecan be derived from a natural source, such as the sperm of salmon oranother species of fish, or the protamine can be produced recombinantly.The term “protamine” includes protamine salts, such as protamine sulfateand protamine hydrochloride.

The term “nucleotidic impurity” refers to any compound derived from apreparation of nucleic acids. In certain embodiments, the nucleotidicimpurity in the protamine sample is associated with the presence of DNA.In some embodiments, the nucleotidic impurity is a single- ordouble-stranded oligonucleotide, a nucleotide, a nucleoside, or anucleobase. Exemplary nucleotidic impurities include, withoutlimitation, adenosine, deoxyadenosine, guanosine, deoxyguanosine,cytidine, deoxycitidine, thymidine, uridine, and methylated orphosphorylated derivatives of the foregoing.

The protamine disrupting agent can be any agent capable of disruptingthe binding between protamine and nucleic acids. In one embodiment, theprotamine disrupting agent is a protease, such as a serine protease.

In a particular embodiment, the protamine-disrupting agent is trypsin.The trypsin can be immobilized, e.g., on a silica stationary phase or onmagnetic beads, or the trypsin can be free in solution. In certainembodiments, the trypsin is inactivated prior to the digestion step. Thetrypsin can be inactivated by heat treatment, or by use of a chemicalinhibitor of trypsin.

In another embodiment, the protamine disrupting agent is a stronglycharged chemical reagent. In one embodiment, the protamine disruptingagent is a positively charged chemical reagent, such as spermine,spermidine, arginine, poly-arginine, or poly-lysine. In anotherembodiment, the protamine disrupting agent is a negatively chargedchemical reagent, such as aspartate or heparin.

In yet another embodiment, the protamine disrupting agent is a physicaltreatment, such as heating or sonication.

In still yet other embodiments, the protamine disrupting agent is asolid phase extraction material, such as a strong cation exchange resin.

In any of the above embodiments, prior to step (c) the first mixture canbe heated under conditions sufficient to inactivate theprotamine-disrupting agent. The presence of the protamine-disruptingagent may interfere with the digestion step. Thus, in certainembodiments, the protamine-disrupting agent may be inactivated byheating. For example, the first mixture can be heated to a temperatureof about 30° C. or more, about 40° C. or more, about 50° C. or more,about 60° C. or more, about 70° C. or more, about 80° C. or more, about90° C. or more, or about 100° C. or more.

In any of the above embodiments, the digestion step comprises subjectingthe first mixture to digestion with an endonuclease, aphosphodiesterase, an alkaline phosphatase, or combinations thereof toprovide the second mixture. The endonuclease, phosphodiesterase, andalkaline phosphatase can be any suitable endonuclease,phosphodiesterase, or alkaline phosphatase. In certain embodiments, theendonuclease is an endonuclease derived from Serratia marcescens. Anexample of a suitable endonuclease is BENZONASE™ endonuclease (EMDMillipore, Billerica, Mass.), which is a genetically engineeredendonuclease derived from Serratia marcescens that is produced andpurified from E. coli strain W3110. In certain embodiments, thephosphodiesterase is phosphodiesterase I.

In one embodiment of the invention, the digestion step comprisessubjecting the first mixture to digestion with an endonuclease, aphosphodiesterase and an alkaline phosphatase. Preferably, the digestionstep comprises subjecting the first mixture to digestion with anendonuclease from Serratia marcescens, phosphodiesterase I, and analkaline phosphatase.

In any of the above embodiments, the step of determining the presence oramount of at least one nucleotidic impurity present in the secondmixture can comprise subjecting the second mixture to a high pressureliquid chromatography to obtain a chromatogram, and comparing at leastone peak obtained in the chromatogram due to the presence of at leastone nucleotidic impurity to at least one peak resulting from achromatogram obtained from a reference standard of the at least onenucleotidic impurity.

In another embodiment, the invention provides a method for analyzing asample of protamine comprising four major peptides corresponding toPeptides 1-4 in FIG. 1 and at least one related impurity, comprising (a)subjecting a solution of the sample of protamine to a high pressureliquid chromatography (HPLC) under conditions suitable to resolve thefour major peptides and at least one related impurity in a chromatogram,(b) determining the areas under the peaks of the four major peptides inthe chromatogram, (c) determining the area under the peak of the atleast one related impurity in the chromatogram, and (d) determining theconcentration of (i) one or more of the four major peptides, (ii) thesum of two or more of the four major peptides, and/or (iii) the at leastone related impurity.

The term “related impurity” refers to any compound which produces a peakon the HPLC chromatogram other than the peaks corresponding to the fourmajor protamine peptides. Related impurities include, withoutlimitation, degradation products of the four major peptides, peptidesderived from a method of isolating protamine from a natural source, andpeptides derived from a process of making protamine recombinantly.Related impurities also include non-peptide compounds detected by theHPLC method, such as nucleic acids and carbohydrates.

Preferably, the determination of the concentration of each of the fourmajor peptides and each related impurity is by area under the peak ofeach major peptide or related impurity as a percentage of the total areaunder all peaks observed in the chromatogram.

In certain embodiments, the HPLC is performed using a C-18chromatography column. In some embodiments, the HPLC column is anXBridge BEH300 C18 Peptide Separation Technology (PST) column whichutilizes a wide-pore (300 Å) trifunctionally bonded ethylene bridgedhybrid (BEH) particle (Waters Corporation, Milford, Mass.).

In some embodiments, the HPLC is performed at a column temperature ofless than 55° C. For example, the HPLC can be performed at a columntemperature of about 25° C., about 30° C., about 35° C., about 40° C.,about 45° C., about 50° C., or any range therein. In certainembodiments, the HPLC is performed at a column temperature of about 40°C.

In certain embodiments, the HPLC is performed using a flow rate of lessthan 1.0 mL/minute. For example, the HPLC can be performed using a flowrate of about 0.9 mL/minute, about 0.8 mL/minute, about 0.7 mL/minute,about 0.6 mL/minute, about 0.5 mL/minute, or any range therein. Incertain embodiments, the HPLC is performed using a flow rate of about0.6 mL/minute.

In certain embodiments, the high pressure liquid chromatography isperformed using a mobile phase A consisting of a monobasic sodiumphosphate aqueous solution and a mobile phase B consisting of a mixtureof a monobasic sodium phosphate aqueous solution and acetonitrile. In apreferred embodiment, the ratio of monobasic sodium phosphate aqueoussolution to acetonitrile in the mobile phase B is about 65:35. Incertain embodiments, the high pressure liquid chromatography isperformed using a gradient consisting of variable amounts of mobilephase A and mobile phase B.

In any embodiment of the invention, a sample solution comprisingprotamine can be a solution of the sample of protamine in aqueous sodiumchloride.

As will be readily understood by one of ordinary skill in the art, thesolvent eluting from the terminus of a chromatography column is referredto as an eluent. The eluent can be monitored using any suitabledetection method. A preferred detection method is determining the UVabsorbance of at least a portion of the eluent as the chromatography isin process. In preferred embodiments, the chromatogram is determined bymeasuring a UV absorbance of the eluent at a wavelength of 214 nm.

In certain preferred embodiments, the peak of the at least one relatedimpurity is present between the peaks corresponding to the majorpeptides corresponding to Peptides 2 and 3 in FIG. 1. In certainpreferred embodiments, two peaks corresponding to at least two relatedimpurities are present between the peaks corresponding to the majorpeptides corresponding to Peptides 2 and 3 in FIG. 1.

In certain embodiments, the determination about the sample of protaminecomprises selecting a batch of protamine as a result of thedetermination.

As shown in the example chromatography and demonstrated in the databelow comparing both the method of the present invention and acomparative method, the method of the present invention demonstratessuperior resolution of major peptides from the related peptides aroundand between the major peptides resulting in a more accurate quantitationof the major peptides and more accurate results for the related peptidesthan the comparative method. Since the present method has betterselectivity than the comparative method, it is easier to integrate allrelated peptides present, especially between major peptides 2 and 3.

In some embodiments, the method for analyzing a sample of protamine rawmaterial results in a determination of the concentration of the sum ofthe four major peptides corresponding to Peptides 1-4 in FIG. 1 which is85% w/w or more, e.g., 85.5% w/w or more, 86.0% w/w or more, 86.5% w/wor more, 87.0% w/w or more, 87.5% w/w or more, 88.0% w/w or more, 88.5%w/w or more, 89.0% w/w or more, 89.5% w/w or more, 90.0% w/w or more,90.5% w/w or more, 91.0% w/w or more, 91.5% w/w or more, or 92.0% w/w ormore. In other embodiments, the method for analyzing a sample ofprotamine raw material results in a determination of the concentrationof the sum of the four major peptides corresponding to Peptides 1-4 inFIG. 1 which is 97.0% w/w or less, e.g., 96.5% w/w or less, 96.0% w/w orless, 95.5% w/w or less, 95.0% w/w or less, 94.5% w/w or less, 94.0% w/wor less, 93.5% w/w or less, 93.0% w/w or less, 92.5% w/w or less, 92.0%w/w or less, 91.5% w/w or less, 90.0% w/w or less, or 89.5% w/w or less.In yet other embodiments, the method for analyzing a sample of protamineraw material results in a determination of the concentration of the sumof the four major peptides corresponding to Peptides 1-4 in FIG. 1 whichis in a range bounded by any of the foregoing values. For example, themethod for analyzing a sample of protamine raw material can result in adetermination of the concentration of the sum of the four major peptidescorresponding to Peptides 1-4 in FIG. 1 which is 86.0%-95.0%,87.5%-90.0% w/w, 88.0%-91.0% w/w, 88.5%-93.0% w/w, 90.0%-92.5% w/w, or87.0%-90.5%.

In some embodiments, the method for analyzing a sample of protaminefinished product, such as a pharmaceutical composition comprisingprotamine, results in a determination of the concentration of the sum ofthe four major peptides corresponding to Peptides 1-4 in FIG. 1 which is76.0% w/w or more, e.g., 77.0% w/w or more, 77.5% w/w or more, 78.0% w/wor more, 78.5% w/w or more, 79.0% w/w or more, 79.5% w/w or more, 80.0%w/w or more, 80.5% w/w or more, 82.0% w/w or more, 83.5% w/w or more,85.0% w/w or more, 86.5% w/w or more, 88.0% w/w or more, or 89.5% w/w ormore. In other embodiments, the method for analyzing a sample ofprotamine finished product results in a determination of theconcentration of the sum of the four major peptides corresponding toPeptides 1-4 in FIG. 1 which is 93.0% w/w or less, e.g., 92.5% w/w orless, 92.0% w/w or less, 91.5% w/w or less, 91.0% w/w or less, 90.5% w/wor less, 90.0% w/w or less, 89.0% w/w or less, 87.5% w/w or less, 86.0%w/w or less, 84.5% w/w or less, 83.0% w/w or less, 81.5% w/w or less, or80.0% w/w or less. In yet other embodiments, the method for analyzing asample of protamine finished product results in a determination of theconcentration of the sum of the four major peptides corresponding toPeptides 1-4 in FIG. 1 which is in a range bounded by any of theforegoing values. For example, the method for analyzing a sample ofprotamine raw material can result in a determination of theconcentration of the sum of the four major peptides corresponding toPeptides 1-4 in FIG. 1 which is 76.0%-92.5%, 77.5%-91.0% w/w,78.0%-83.0% w/w, 79.0%-86.0% w/w, 80.5%-89.0% w/w, or 85.0%-91.0%.

The methods comprise chromatographically separating a sample comprisingprotamine using liquid chromatography. The invention is not limited toany particular manner of performing liquid chromatography. In general,the chromatographic separation step includes using at least one liquidchromatography (LC) column. In some embodiments, multiple LC columns areused, such as two or more, or three or more, or four or more LC columns.In some such embodiments two, three, four, five, six, eight, or ten LCcolumns are used. In some such embodiments, two or more of these LCcolumns are arranged parallel to each other.

The invention is not limited to any particular types of columns. Anycolumn suitable for the separation of protamine can be used. In someembodiments, one or more analytical columns are used. In some suchembodiments, one or more reverse phase columns are used. In someembodiments, the method employs two or more reverse phase columns inparallel.

Further, the invention is not limited to any particular mobile phase.Any suitable mobile phase or phases can be used, as long as the mobilephase or phases are suitable for use with a particular LC column and forchromatographically separating the protamine sulfate in the LC column.In some embodiments, the mobile phase or phases are a polar solventsystem or systems. The polar solvent system or systems can include oneor more polar solvents, including but not limited to water, methanol,acetonitrile, or a mixture of two or more of the foregoing. The polarsolvent system can include one or more aqueous buffer solutions. Forexample, the polar solvent system can comprise, consist essentially of,or consist of an aqueous solution of one of more buffers. In otherembodiments, the aqueous solution of one or more buffers furtherincludes one or more polar solvents in addition to water. In someembodiments, the mobile phase employs a gradient obtained by mixing twoor more solvents, such that the relative ratios of the two or moresolvents are varied over time.

As noted above, two or more LC columns (e.g., reverse phase columns) canbe used in parallel, e.g., to improve throughput. In some suchembodiments, a protamine sulfate sample e.g., a partially purifiedsample or a crude mixture) is introduced to the two or more LC columnsat different times. In some embodiments, the introduction of the testsample to the two or more LC columns is staggered, meaning that there isa pre-determined time interval separating the introduction of sample totwo or more LC columns. Appropriate time intervals can be selected basedon various factors, including the elution time, column chemistries, andthe potential need to avoid interfering with the analysis of theprotamine sulfate eluted from one or more of the other LC columns.

In some embodiments of the invention, another LC column can be placed inseries with another column. For example, in some embodiments, suitableguard columns can be employed. Those of skill in the art are able toselect appropriate guard columns for use in the present methods. In someembodiments, a guard column is placed in parallel with another LCcolumn, and both the guard column and the LC column are reverse phasecolumns. Such series of two or more columns can also be arranged inparallel, such that there are two or more series of columns operating inparallel, where each series contains two or more columns.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates the use of high performance liquidchromatographic (HPLC) analysis for the determination of nucleotidicimpurities in Protamine Sulfate, USP raw material by HPLC.

Summaries of the analytical solutions and HPLC parameters used toanalyze samples for the presence or amount of at least one nucleotidiccomponent are provided in the following tables.

Analytical Solutions

Mobile Phase 20 mM ammonium acetate pH 4.1 A (MP A) Mobile PhaseAcetonitrile (ACN) B (MP B) Standard The Standard Solution containsapproximately Solution 0.125 μg/mL of USP Adenosine Reference Standard(RS) in water. System The System Sensitivity Solution containsapproximately Sensitivity 0.05 μg/mL of Adenosine RS in water. SolutionNucleoside The Nucleoside Identification Solution containsIdentification approximately 2.5 μg/mL uridine, guanosine, cytidine,Solution thymidine, 2′-deoxyadenosine, 2′-deoxyguanosine,2′-deoxycytidine, and 5-methyl-2′-deoxycytidine in water. Reaction 20 mMmagnesium chloride (MgCl2), 20 mM Tris, Buffer 100 mM sodium chloride(NaCl) in water pH 7.9 Phospho- 0.1 U/μL in 1:1 Reaction Buffer:Glycerol diesterase I (PDE I) Solution: Enzyme Digest 50 U/mL BenzonaseEndonuclease, Solution 44.4 U/mL Alkaline Phosphatase, and 2.5 U/mL PDEI in Reaction Buffer Diluent 64 mM Sodium Bicarbonate pH 8.5 Sample TheSample Solution contains approximately 0.5 mg/mL Solution protaminesulfate. Positive The Positive Control Sample Solution contains Controlapproximately 0.5 mg/mL protamine sulfate and Sample 0.5 μg/mL of DNA.Solution

HPLC Parameters

Column Phenomenex Synergi Fusion-RP 80 Å, 4.6 × 150 mm, 4 μm, PartNumber 00F-4424-E0 Column 25 ± 3° C. Temperature Flow Rate 0.9 mL/minAutosampler 37 ± 2° C. Temperature Injection Volume 100 μL Detector UVat 260 nm Run Time About 30 minutes Time, min % MPA % MPB GradientProfile 0 98 2 5 98 2 15 80 20 20 80 20 20.1 98 2 30 98 2 SystemSuitability 1. The signal-to-noise ratio (S/N) of the Adenosine peak inthe System Sensitivity Solution is NLT 10. 2. The percent relativestandard deviation (% RSD) of the Adenosine peak area responses in six(6) replicate injections of Reference Solution is not more than (NMT)10%. 3. The resolution between 2′-deoxycytidine and the uridine peak inthe Nucleoside Identification Solution is NLT 1.5. 4. All fournucleoside peaks for DNA: 2′-deoxy- adenosine; 2′-deoxyguanosine;2′-deoxycytidine; and thymidine, must be present in Positive ControlSample Solution.

The results of the analysis of the Nucleoside Identification Solutionand Protamine Sulfate, USP Raw Material obtained from three differentsources are shown in FIG. 2.

The results of this example demonstrate a method for analyzing a samplefor the presence or amount of at least one nucleotidic component byHPLC.

Example 2

This example demonstrates a method for analyzing a protamine sample forthe presence or amount of at least one nucleotidic impurity according toan embodiment of the invention.

Equipment

1. HPLC system equipped with a temperature controlled columncompartment, a heated autosampler, a single wavelength UV detector,sample heater, and a suitable data acquisition system2. HPLC column: Phenomenex Synergi Fusion-RP 80 Å, 4.6×150 mm, 4 μm,Part Number 00F-4424-E0

3. HPLC Guard Column: Phenomenex Security Guard Cartridge Kit, PartNumber KJ0-4282 4. HPLC Guard Cartridge: Phenomenex Security GuardCartridge Fusion-RP 4 X 3.0 mm ID Part Number AJ0-7557

5. 3004 HPLC vial, Agilent Part #5188-6591, or equivalent6. Blue Screw Caps for HPLC vial, Agilent Part #5182-0717, or equivalent7. 1000 μL Eppendorf vial, Fisher Part#02-681-284 or equivalent8. Calibrated analytical balance9. Calibrated pH meter10. Calibrated micropipettes11. Heat block capable of maintaining 100±3° C.

12. Calibrated Thermometer

13. Oven capable of maintaining 37° C.±3° C.14. Mini Centrifuge, Fisher Scientific, Cat #05-090-100, or equivalent15. Nutating Mixer, Fisher Scientific, Cat #05-450-213, or equivalent

16. Calibrated Timers 17. Sonicator 18. Freezer (−15 to −25° C.) 19.Refrigerator (2 to 8° C.)

20. Class A volumetric glassware21. Other standard laboratory glassware and equipment

Materials

1. Acetonitrile, HPLC grade (ACN)2. Water, HPLC grade or Purified Water, USP3. Ammonium Acetate, ACS grade, or equivalent4. Glacial Acetic Acid, ACS grade, or equivalent5. Magnesium Chloride 6-Hydrate, USP grade, or equivalent6. Tris(hydroxymethyl)amino methane, ACS grade (tris), or equivalent7. Sodium Chloride, ACS grade, or equivalent8. 1N Hydrochloric Acid, NIST grade9. 1N Sodium Hydroxide, NIST grade10. Glycerol, ACS grade, or equivalent

11. Benzonase, 99%, 25 U/μL EMD Part #70664-3 12. Phosphodiesterase I100 U/vial, Worthington Biochemical Part # LS003926 (PDE I) 13. AlkalinePhosphatase, Sigma Part # P7923-10kU (AP)

14. Protamine Sulfate, USP raw material

15. USP Adenosine Reference Standard (RS), Catalog #1012123

16. Uridine Reference Standard (RS), (Sigma Product # U6381), orequivalent17. Guanosine RS, (Acros Product #41113), or equivalent18. Cytidine RS, (Acros Product #11181), or equivalent19. Thymidine RS, (Acros Product #22674), or equivalent20. 2′-Deoxyadenosine Hydrate RS, (Acros Product #15499), or equivalent21. 2′-Deoxyguanosine Hydrate RS, (Acros Product #22673), or equivalent22. 2′-Deoxycytidine RS, (Sigma Product # D3897), or equivalent23. 5-Methyl-2′-Deoxycytidine RS, (Berry Product # PY 7635), orequivalent24. Deoxyribonucleic acid sodium salt (DNA) from calf thymus, (SigmaProduct # D1501)25. Trypsin from porcine pancreas, (Sigma Product # T6567)

Preparations

Mobile Phase a (20 mM Ammonium Acetate pH 4.1)

Accurately weigh and transfer about 3.08 g ammonium acetate in a 2-Lflask, add 2000 mL water, and mix until dissolved. Adjust pH to 4.1±0.1with glacial acetic acid. Degas for about 2 minutes under vacuum withsonication. Prepare a proportionally larger quantity if necessary.

Mobile Phase B (100% Acn)

Transfer 2000 mL ACN into a 2-L flask. Degas for about 1 minute undervacuum with sonication.

Nucleoside Identification Solution

Accurately weigh and transfer about 25 mg of Uridine RS, Guanosine RS,Cytidine RS, Thymidine RS, 2′-Deoxyadenosine RS, 2′-Deoxyguanosine RS,2′-Deoxycytidine RS, and 5-Methyl-2′-Deoxycytidine RS into a 200 mLvolumetric flask, add approximately 185 mL of water, dissolve withsonication and vortexing (if necessary), dilute to volume, and mix.Transfer 2.0 mL of this solution into a 100-mL volumetric flask, diluteto volume with water, and mix. This solution contains approximately 2.5μg/mL of each nucleoside.

Adenosine Stock Standard Solution

Accurately weigh and transfer about 25 mg of USP Adenosine RS into a100-mL volumetric flask, add approximately 85 mL of water, and dissolvewith sonication and vortexing (if necessary). Dilute to volume withwater and mix. Transfer 10.0 mL of this solution into a 100-mLvolumetric flask, dilute to volume with water and mix. This solutioncontains approximately 25 μg/mL adenosine.

Standard Solution

Transfer 1.0 mL of Adenosine Stock Standard Solution into a 200-mLvolumetric flask, dilute to volume with water, and mix. This solutioncontains approximately 0.125 μg/mL of adenosine.

System Sensitivity Solution

Transfer 4.0 mL Standard Solution into a 10-mL volumetric flask, diluteto volume with water, and mix. This solution contains approximately 0.05μg/mL adenosine.

Reaction Buffer

Accurately weigh and transfer 410 mg magnesium chloride hexahydrate, 240mg tris, and 580 mg sodium chloride into a 100-mL volumetric flask,dissolve in 75 mL water, and mix. Adjust pH to 7.9±0.1 with 1Nhydrochloric acid. Dilute to volume with water and mix. Storerefrigerated at 5±3° C.

PDE I Diluent (1:1 Reaction Buffer:Glycerol)

Transfer 5.0 mL glycerol and 5.0 mL Reaction Buffer into a 20-mLvolumetric flask and vortex to mix.

PDE I Solution

Using a small spatula carefully transfer the contents of 1 vialPhosphodiesterase I (PDE I) into a 1000 μL Eppendorf vial andreconstitute with 1000 μL PDE I Diluent. This solution has aconcentration of approximately 0.1 U/μL.

Enzyme Digest Solution

Add 10 μL Benzonase, 222 U Alkaline Phosphatase (AP), and 125 μL PDE ISolution to 5.0 mL Reaction Buffer. This solution can be proportionallyadjusted to prepare the desired amount. This solution contains 50 U/mLBenzonase, 44.4 U/mL AP, and 2.5 U/mL PDE I. Calculate the volume of APto be used as follows using the C of A per Equation 1:

$\begin{matrix}{{\mu \; L\; {AP}} = {\frac{{Units}\mspace{14mu} {AP}}{\left( {{{Units}/{mg}}\mspace{14mu} {Protein}} \right) \times \left( {{mg}\mspace{14mu} {{Protein}/{mL}}} \right)} \times \frac{1000\mspace{11mu} \mu \; L}{mL}}} & (1)\end{matrix}$

where,

-   Units AP=Units of AP needed to prepare Enzyme Digest Solution-   Units/mg Protein=Enzymatic activity of AP from C of A in Units/mg    Protein-   mg Protein/mL=Protein by Biuret with TCA Precipitation from C of A    in mg protein/mL

Diluent (64 mM Sodium Bicarbonate pH 8.5)

Accurately weigh and transfer 2.70 g sodium bicarbonate into a 500-mLvolumetric flask, dissolve in 450 mL water, and mix. Adjust pH to8.5±0.1 with 1N hydrochloric acid or 1N sodium hydroxide. Dilute tovolume with water and mix. Store refrigerated at 5±3° C.

1 mM Hydrochloric (HCl) Acid

Transfer 1.0 mL 0.1N hydrochloric acid into a 100-mL volumetric flask,dilute to volume with water and mix.

Blank Solution

Listed below are the steps necessary to prepare Blank Solution.

1. Reconstitute 20 μg vial of trypsin with 20 μL 1 mM HCl. Transfer 100μL of the Diluent into a trypsin reagent vial. Digest for not less than(NLT) 60 minutes in vial with mixing at 37±2° C. using an oven.2. Using a heat block inactivate trypsin for NLT 30 minutes at 100±3° C.Flash cool at −20° C. for 2-3 minutes. Spin reagent vial in minicentrifuge for approximately 30 seconds. Confirm that sample solutionaccumulates at the bottom of the reagent vial.3. Transfer entire reaction mixture (120 μL) into a 300 μL HPLC vial,add 80 μL Enzyme Digest Solution and mix with the micropipette. IncubateNLT 60 minutes in the autosampler at 37° C. before injection.

Sample Solution

Listed below are the steps necessary to prepare Sample Solution.

1. Accurately weigh and transfer about 20 mg Protamine Sulfate, USP rawmaterial into a 20-mL volumetric flask, dilute to volume with Diluent,and mix.2. Reconstitute 20 μg vial of trypsin with 20 μL 1 mM HCl. Transfer 100μL of the 1 mg/mL Protamine Sulfate solution into a trypsin reagentvial. Digest for NLT 60 minutes in vial with mixing at 37±2° C. using anoven.

Using a heat block inactivate trypsin for NLT 30 minutes at 100±3° C.Flash cool at −20° C. for 2-3 minutes. Spin reagent vial in minicentrifuge for approximately 30 seconds. Confirm that sample solutionaccumulates at the bottom of the reagent vial.

4. Transfer entire reaction mixture (120 μL) into a 300-4 HPLC vial, add80 μL Enzyme Digest Solution and mix with the micropipette. Incubate NLT60 minutes in the autosampler at 37° C. before injection. This solutioncontains approximately 0.5 mg/mL protamine sulfate.

Positive Control Sample Solution (0.1% DNA)

Listed below are the steps necessary to prepare Sample Solution.

1. Accurately weigh and transfer about 20 mg Protamine Sulfate, USP rawmaterial into a 20-mL volumetric flask, dilute to volume with Diluent,and mix.2. Accurately weigh and transfer about 1.1 mg of DNA into a 100 mLvolumetric flask, dilute to volume with water, and mix to dissolve.Correct for purity [from Certificate of Analysis(COA)] of the DNA whileweighing. % Purity=A260 Unit/mg solid absorbance value (from COA÷20)Note: Do not sonicate or vortex DNA to dissolve.3. Reconstitute 20 μg vial of trypsin with 20 μL 1 mM HCl. Transfer 90μL of the 1 mg/mL Protamine Sulfate solution and 10 μL of the 10 μg/mLDNA solution into a trypsin reagent vial. Digest for NLT 60 minutes invial with mixing at 37±1° C. using an oven/chamber.4. Using a heat block inactivate trypsin for NLT 30 minutes at 100±3° C.Flash cool at −20° C. for 2-3 minutes. Spin reagent vial in minicentrifuge for approximately 30 seconds. Confirm that sample solutionaccumulates at the bottom of the reagent vial.5. Transfer entire reaction mixture (120 μL) into a 300-μL HPLC vial,add 80 μL Enzyme Digest Solution and mix with the micropipette. IncubateNLT 60 minutes in the autosampler at 37° C. before injection. Thissolution contains approximately 0.5 mg/mL protamine sulfate.

DNA Control Sample Solution (0.1% DNA)

Listed below are the steps necessary to prepare Sample Solution.

1. Accurately weigh and transfer about 1.1 mg of DNA into a 100 mLvolumetric flask, dilute to volume with water, and mix to dissolve.Correct for purity (from COA) of the DNA while weighing.% Purity=A260 Unit/mg solid absorbance value (from COA÷20)Note: Do not sonicate or vortex DNA to dissolve.2. Reconstitute 20 μg vial of trypsin with 20 μL 1 mM HCl. Transfer 90μL of Diluent and 10 μL of the 10 μg/mL DNA solution into a trypsinreagent vial. Digest for NLT 60 minutes in vial with mixing at 37±1° C.using an oven/chamber.3. Using a heat block inactivate trypsin for NLT 30 minutes at 100±3° C.Flash cool at −20° C. for 2-3 minutes. Spin reagent vial in minicentrifuge for approximately 30 seconds. Confirm that sample solutionaccumulates at the bottom of the reagent vial.4. Transfer entire reaction mixture (120 μL) into a 300-4 HPLC vial, add80 μL Enzyme Digest Solution and mix with the micropipette. Incubate NLT60 minutes in the autosampler at 37° C. before injection. This solutioncontains approximately 0.5 mg/mL protamine sulfate.

Chromatographic System

HPLC Parameters

-   -   Column: Phenomenex Synergi Fusion-RP 80 Å, 4.6×150 mm, 4 μm,        Part Number 00F-4424-E0    -   Guard Column: Phenomenex Security Guard Cartridge Kit, Part        Number KJ0-4282 Phenomenex Security Guard Cartridge, Synergi        Fusion-RP 4×3.0 mm, Part Number AJ0-7557    -   Column Temperature: 25±3° C.    -   Mobile Phase A (MP A): 20 mM ammonium acetate pH 4.1    -   Mobile Phase B (MP B): Acetonitrile    -   Gradient:

Time % MPA % MPB 0 98 2 5.00 98 2 15.00 80 20 20.00 80 20 20.10 98 230.00 98 2

-   -   Flow Rate: 0.9 mL/min.    -   Injection Volume: 100 μL    -   Detector: 260 nm    -   Needle Wash: Water    -   Autosampler Temperature: 37±1° C.    -   Run Time: 30 minutes

Robustness Range

Parameter Range Change Column Temperature 20-30° C. ±5° C. Mobile PhaseA pH 4.0-4.2 ±0.1 pH Diluent pH 8.4-8.6 ±0.1 pH Digest Time (minutes)45-75 ±15 Reaction Buffer pH 7.8-8.0 ±0.1 pH Inactivation Time (minutes)20-40 ±10 Incubation Temperature 36-38° C. ±1° C.

System Suitability

1. Inject six (6) replicates of the Standard Solution and record thechromatograms. The percent relative standard deviation (% RSD) of theareas of the adenosine peak is not more than (NMT) 10%.2. The resolution between the 2′-deoxycytidine peak and the Uridine peakis NLT 1.5 for the injection of the Nucleoside Identification Solution.3. The signal-to-noise ratio (S/N) of the adenosine peak in the SystemSensitivity Solution is NLT 10.4. All four nucleoside peaks for DNA-2′ deoxycytidine, 2′deoxyguanosine, Thymidine, 2′ deoxyadenosine must be present in thePositive Control Sample Preparation.

Procedure

Separately inject 100 μL of the water, Blank Solution, SystemSensitivity Solution, Nucleoside Identification Solution, StandardSolution, Sample Solution, and Positive Control Sample Preparation andrecord the chromatograms.

Nucleotidic Impurity Calculations

Area Reject Value (Q)

Calculate the area reject value, Q, which corresponds to a S/N of 10.The Q value is calculated using Equation (2) and will be used as thecut-off area for considering individual peaks.

Q=(10×A _(sss))/S/N  (2)

where:A_(sss)=Peak area of adenosine in the System Sensitivity SolutionS/N=Signal-to-noise ratio of the adenosine peak in the SystemSensitivity Solution

Standard Solution

Calculate the concentration of adenosine, in mg/mL, using Equation (3).

C _(std) =W _(std) /DF  (3)

where:C_(std)=Concentration of adenosine in the Standard Solution, in mg/mL

W_(std)=Weight of USP Adenosine RS, in mg DF=200,000 mL (DilutionFactor)

Nucleotidic Impurities

Calculate the % Nucleotidic Impurities using Equation (4):

$\begin{matrix}{{\% \mspace{14mu} {NUCLEOTIDIC}\mspace{14mu} {{IMP}.}} = {\sum\; {\left( {\frac{C_{STD}}{A_{STD}} \times A_{i} \times \frac{{MW}_{RATIO}}{{RRF}_{1}}} \right) \times \frac{DF}{W_{SAMPLE}} \times 100\%}}} & (4)\end{matrix}$

where:

-   -   C_(std)=Concentration of adenosine in the Standard Solution, in        mg/mL    -   A_(std)=Average peak area (n=6) of adenosine in the Standard        Solution    -   A_(i)=Peak area of each impurity above Q in the Sample Solution    -   RRF_(i)=For known impurities see Table I otherwise use 1.00    -   MW_(ratio)=For known impurities see Table I otherwise use 1.00    -   DF=40 mL (Dilution Factor)    -   W_(sample)=Sample weight of Protamine Sulfate, USP, in mg

Relative Retention Times (RT), Relative Response Factors (RRF), andMolecular Weight (MW) Ratios for Known Impurities

RRT Nucleoside (min) RRF MW_(ratio) Cytidine 0.28 0.53 1.25482′-Deoxycytidine 0.35 0.56 1.2727 Uridine 0.39 0.75 1.2537 5-Methyl-2′-0.55 0.25 1.2569 Deoxycytidine HCl Guanosine 0.72 0.74 1.21882′-Deoxyguanosine 0.87 0.83 1.2319 Thymidine 0.93 0.68 1.2558 Adenosine1.00 1.00 1.2319 2′-Deoxyadenosine 1.04 1.09 1.2466

This example demonstrates a method of analyzing a protamine sample forthe presence or amount of at least one nucleotidic impurity according toan embodiment of the invention.

Example 3

This example demonstrates a method for analyzing a sample of protaminesulfate comprising four major peptides and related impurities by an HPLCmethod.

Equipment

1. An HPLC system (Agilent 1100 or 1200, or Dionex Ultimate 3000)equipped with a temperature controlled column compartment, temperaturecontrolled autosampler, variable wavelength UV/VIS detector, and asuitable data acquisition system2. HPLC column: Waters Xbridge BEH 300, C-18 (3.5 μm, 4.6 mm×150 mm)P/No 1860036133. For Agilent 1100 or 1200 only, Thermal Column Heat Block, P/NoG1316-600014. Analytical balance, calibrated5. pH meter, calibrated6. Vortex mixer

7. Sonicator

8. Hygrometer, calibrated9. Class A volumetric glassware10. Other standard laboratory glassware and equipment

Materials

1. Acetonitrile, HPLC grade2. Purified Water, USP or HPLC grade, or equivalent3. Sodium phosphate monobasic monohydrate, HPLC grade, or equivalent4. Hydrochloric acid, ACS grade5. Sodium chloride, ACS grade

6. Protamine Sulfate, USP Raw Material 7. Protamine Sulfate Injection,USP Finished Product

8. Nitrogen gas, Ultra High Purity Grade

Preparations

Mobile Phase A

Accurately weigh and transfer about 22.1 grams of sodium phosphatemonobasic monohydrate into a 2 L container containing 2 L of water andmix. Adjust pH to about 1.9±0.2 with hydrochloric acid. Degas undervacuum with sonication for about 5 minutes. Prepare proportionatelylarger quantities as needed.

Mobile Phase B

Transfer 650 mL of Mobile Phase A and 350 mL of acetonitrile into asuitable container and mix. Degas under vacuum with sonication for about3 minutes. Prepare proportionately larger quantities as needed.

Diluent

Accurately weigh and transfer about 9 grams of sodium chloride into a1-L suitable container. Transfer 1000 mL of water into the container andmix. Prepare proportionately larger quantities as needed.

Reference Solution

Accurately weigh and transfer about 100 mg of Protamine Sulfate, USP rawmaterial into a 10-mL volumetric flask, dissolve and dilute to volumewith Diluent with alternating vortexing and sonication. The ReferenceSolution contains approximately 10 mg/mL of protamine sulfate. Note:weigh protamine sulfate raw material under nitrogen with less than 20%relative humidity.

System Sensitivity Solution

Pipette 5.0 mL of the Reference Solution into a 25-mL volumetric flask,dilute to volume with Diluent, and mix. Pipette 3.0 mL of this solutioninto a 100-mL volumetric flask, dilute to volume with Diluent, and mix.The System Sensitivity Solution contains approximately 60 μg/mL ofprotamine sulfate.

Raw Material Sample Solution

Accurately weigh and transfer about 100 mg of Protamine Sulfate, USP rawmaterial into a 10-mL volumetric flask, dissolve and dilute to volumewith Diluent with alternating vortexing and sonicating. The Raw MaterialSample Solution contains approximately 10 mg/mL of protamine sulfate.Note: weigh protamine sulfate raw material under nitrogen with less than20% relative humidity.

Finished Product Sample Solution

The Finished Product Sample Solution has a concentration ofapproximately 10 mg/mL protamine sulfate. Transfer appropriate aliquotof finished product sample into a HPLC vial and inject sample neat.

Chromatographic System

HPLC Parameters

-   Mobile Phase A (MP A): 80 mM sodium phosphate monobasic in water, pH    1.9±0.2-   Mobile Phase B (MP B): 80 mM sodium phosphate monobasic pH 1.9±0.2    in water:acetonitrile (65:35)-   Gradient:

Time (min) MP A MP B 0.0 96.1 3.9 30.0 91.5 8.5 40.0 91.5 8.5 41.0 1.099.0 51.0 1.0 99.0 52.0 96.1 3.9 70.0 96.1 3.9

-   Column: Waters XBridge BEH 300, C-18, 3.5 μm, 4.6 mm×150 mm, P/No    186003613-   Column Temperature: 40−1+2° C.-   Flow Rate: 0.6 mL/min.-   Injection Volume: 10 μL-   Autosampler Temperature: 25±3° C.-   Detection: UV at 214 nm-   Run Time: 70 minutes-   Needle Wash: Acetonitrile:Water 50:50-   Separation Mode: Gradient

Robustness Conditions

Condition Range Change Column Temperature (40° C.) 39 to 42° C. −1 + 2°C. Mobile Phase A pH (1.9) 1.7 to 2.1 ±0.2 Mobile Phase A 75 to 85 mM ±5mM Buffer Concentration (80 mM)

System Suitability

Adjust sequence to inject the Reference Solution before the SystemSensitivity Solution.

1. Chromatograph the System Sensitivity Solution. The signal-to-noiseratio (S/N) of the protamine sulfate peptide #2 peak is not less than(NLT) 10.2. Chromatograph the Reference Solution. The percent relative standarddeviation (% RSD) of the protamine sulfate peak area sum of four majorpeptide peak responses in five (5) replicate injections is not more than(NMT) 2.0%.3. In the first Reference Solution injection, the resolution betweenpeaks of peptide 1 and peptide 2 is not less than (NLT) 2.5. Theresolution between peaks of peptide #2 and peptide #3 is NLT 1.8. Theresolution between peaks of peptide #3 and peptide #4 is not less thanNLT 2.5.4. In the first Reference Solution injection, the column efficiency isNLT 4,000 theoretical plates for peptide #1, NLT 1,500 theoreticalplates for peptide #2, NLT 3,500 theoretical plates for peptide #3, andNLT 4,500 theoretical plates for peptide #4.

Procedure

Separately inject 10 μL of Diluent, Reference Solution, SystemSensitivity Solution, and Raw Material Sample Solution and/or FinishedProduct Sample Solution into the HPLC and record the chromatograms.

Identification

HPLC Retention Times

The chromatogram of the Raw Material Sample Solution or Finished ProductSample Solution exhibits four (4) major peptide peaks for protaminesulfate, the retention time of which corresponds to that exhibited byprotamine sulfate in the chromatogram of Reference Solution and meetsthe following criterion:

0.95<(R _(u) /R _(s))<1.05  (1)

where,

-   -   R_(u)=Retention time of each major peptide peaks 1-4 in the Raw        Material Sample Solution or Finished Product Sample Solution    -   R_(s)=Retention time of each major peptide peaks 1-4 in the        Reference Solution

This example demonstrates a method of analyzing a protamine samplecomprising four peptides and at least one impurity according to anembodiment of the invention.

Example 4

This example demonstrates a method for analyzing a sample of protaminesulfate comprising four major peptides corresponding to Peptides 1-4 inFIG. 1 and related impurities by the inventive method and a comparativemethod.

Method Summary for an Embodiment of the Inventive Method

The summaries of the HPLC preparations and parameters for an embodimentof the method of the invention are provided in Tables I and II.

TABLE I Preparations for an Embodiment of the Inventive Method MobilePhase A (MP A) 80 mM sodium phosphate monobasic monohydrate, pH 1.9 ±0.2 (Buffer) Mobile Phase B (MP B) 65:35 Buffer:acetonitrile Diluent0.9% sodium chloride in water Reference Solution The Reference Solutioncontains approximately 10 mg/mL of protamine sulfate raw material inDiluent. System Sensitivity Solution The System Sensitivity Solutioncontains approximately 60 μg/mL of Protamine Sulfate Reference Solution(0.2% of abundance of Peptide # 2 at 32%) in Diluent. Raw Material (RM)Raw Material Sample Solution contains Sample Solutions approximately 10mg/mL protamine sulfate in Diluent. Finished Product (FP) FinishedProduct Sample Solution contains Sample Solutions approximately 10 mg/mLprotamine sulfate (neat injection).

TABLE II HPLC Parameters for an Embodiment of the Inventive MethodColumn Waters X-Bridge BEH 300 C-18, 3.5 μm, 4.6 mm × 150 mm, P/No186003613 Column Temperature 40° C. (−1° C. + 2.0° C.) Flow Rate 0.6mL/min. Autosampler Temperature 25 ± 3° C. Injection Volume 10 μLDetector UV at 214 nm Run Time About 70 minutes Time (minutes) % MP A %MP B Gradient Profile 0 96.1 3.9 30 91.5 8.5 40 91.5 8.5 41 1.0 99.0 511.0 99.0 52 96.1 3.9 70 96.1 3.9

Method Summary for the Comparative Method

The summaries of the preparations and HPLC parameters for thecomparative method are provided in Tables III and Iv.

TABLE III Preparations for Comparative Method Mobile Phase A (MP A) 300mM sodium phosphate, pH 1.8 (Buffer) Mobile Phase B (MP B) 93.5:6.5Buffer:acetonitrile Diluent 0.01M HCl in water Standard Solution TheStandard Solution contains approximately 0.5 mg/mL of protamine sulfateraw material in Diluent. System Sensitivity Solution Not Applicable RawMaterial (RM) Raw Material Sample Solution contains Sample Solutionsapproximately 0.5 mg/mL protamine sulfate in Diluent. Finished Product(FP) Finished Product Sample Solution contains Sample Solutionsapproximately 0.5 mg/mL protamine sulfate.

TABLE IV HPLC Parameters for Comparative Method Column PhenomonexJupiter C-18, 5.0 μm, 4.6 mm × 250 mm, P/No OOG-4053-EO ColumnTemperature 55° C. Flow Rate 1.0 mL/min. Autosampler Temperature 25 ± 3°C. Injection Volume 100 μL Detector UV at 214 nm Run Time About 45minutes Time (minutes) % MP A % MP B Gradient Profile¹ 0 85 15 15 55 4525 55 45 30 85 15 45 85 15 ¹Note: Gradient profile was modified toaddress problems with late eluting peaks that were carrying over intothe next sample injection. Therefore, run time was extended toequilibrate the column back to the initial conditions.

The percent total major peptide for raw material analyzed per theinventive method ranged from 88.84 to 94.47 and per the comparativemethod ranged from 90.26 to 95.82. The largest related peptide for rawmaterial analyzed in the inventive method was 3.2% and in thecomparative method was 2.3%. The percent total related peptide for rawmaterial analyzed per the inventive method ranged from 5.4 to 12.3 andper the comparative method ranged from 4.1 to 9.7.

The percent total major peptide for finished product analyzed per theinventive method ranged from 77.35 to 91.07 and per the comparativemethod ranged from 80.06 to 92.12. The largest related peptide forfinished product analyzed per the inventive method was 3.61% and per thecomparative method was 5.67%. The percent total related peptide forfinished product analyzed per the inventive method ranged from 8.9 to22.5 and per the comparative method ranged from 7.8 to 19.9.

A representative full scale chromatogram of Reference Solution forprotamine sulfate analyzed by the method of the present invention isprovided in FIG. 1, and a corresponding expanded scale chromatogram (10to 38 minutes) is provided in FIG. 3. A representative full scalechromatogram of Reference Solution for protamine sulfate analyzed by thecomparative method is provided in FIG. 4, and a corresponding expandedscale chromatogram (10 to 38 minutes) is provided in FIG. 5. Expandedscale chromatograms (10 to 38 minutes) of a sample of protamine sulfatethat were obtained by the present invention and the comparative methodare provided in FIG. 6A and FIG. 6B, respectively.

The results of this example demonstrate that the method of analyzingprotamine according to the invention provides enhanced sensitivity forthe detection of impurities, particularly two impurities which produceHPLC peaks between the HPLC peaks corresponding to protamine majorpeptides 2 and 3, as compared to the comparative method.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method for analyzing a protamine sample for the presence or amountof at least one nucleotidic impurity, comprising the steps of: (a)providing a protamine sample, (b) subjecting the sample of protamine todisruption with a protamine-disrupting agent to provide a first mixture,(c) subjecting the first mixture to a digestion step to provide a secondmixture, and (d) determining the presence or amount of the at least onenucleotidic impurity present in the second mixture.
 2. The method ofclaim 1, wherein the protamine-disrupting agent is trypsin.
 3. Themethod of claim 2, comprising, prior to step (c), heating the firstmixture under conditions sufficient to inactivate theprotamine-disrupting agent.
 4. The method of claim 1, wherein thedigestion step comprises subjecting the first mixture to digestion withan endonuclease, a phosphodiesterase, an alkaline phosphatase, orcombinations thereof to provide the second mixture.
 5. The method ofclaim 1, wherein the determination comprises subjecting the secondmixture to a high pressure liquid chromatography to obtain achromatogram, and comparing a peak obtained in the chromatogramresulting from the at least one nucleotidic impurity to a peak resultingfrom a reference standard of the at least one nucleotidic impurity. 6.The method of claim 5, wherein the determination is of the presence ofthe at least one nucleotidic impurity.
 7. The method of claim 5, whereinthe determination is of the amount of the at least one nucleotidicimpurity.
 8. The method of claim 5, wherein the determination furthercomprises selecting a batch as a result of the determination based uponcomparison to the reference standard.
 9. The method of claim 1, whereinthe at least one nucleotidic impurity is a nucleoside.
 10. The method ofclaim 1, wherein the protamine is protamine sulfate.
 11. A method foranalyzing a sample of protamine comprising four major peptidescorresponding to Peptides 1-4 in FIG. 1 and at least one relatedimpurity, comprising: (a) subjecting a solution of the sample ofprotamine to a high pressure liquid chromatography (HPLC) underconditions suitable to resolve the four major peptides and at least onerelated impurity in a chromatogram, (b) determining the areas under thepeaks of the four major peptides in the chromatogram, (c) determiningthe area under the peak of the at least one related impurity in thechromatogram, and (d) determining the concentration of (i) one or moreof the four major peptides, (ii) the sum of two or more of the fourmajor peptides, and/or (iii) the at least one related impurity.
 12. Themethod of claim 11, wherein the HPLC is performed using a mobile phase Aconsisting of a monobasic sodium phosphate aqueous solution and a mobilephase B consisting of a mixture of a monobasic sodium phosphate aqueoussolution and acetonitrile.
 13. The method of claim 12, wherein the ratioof monobasic sodium phosphate aqueous solution to acetonitrile in themobile phase B is about 65:35.
 14. The method of claim 12, wherein theHPLC is performed using a gradient consisting of variable amounts ofmobile phase A and mobile phase B.
 15. The method of claim 11, whereinthe HPLC is performed at column temperature of about 40° C.
 16. Themethod of claim 11, further comprising after step (d) comparing theconcentration of (i) one or more of the four major peptides, (ii) thesum or two or more of the four major peptides and/or (iii) the at onerelated impurity to a reference standard for protamine.
 17. The methodof claim 11, wherein the peak corresponding to the at least one relatedimpurity is present between the peaks corresponding to the majorpeptides corresponding to Peptides 2 and 3 in FIG.
 1. 18. The method ofclaim 17, wherein two peaks corresponding to at least two relatedimpurities are present between the peaks corresponding to the majorpeptides corresponding to Peptides 2 and 3 in FIG.
 1. 19. The method ofclaim 11, wherein the determination about the sample of protaminecomprises selecting a batch of protamine as a result of thedetermination.
 20. The method of claim 11, wherein the protamine isprotamine sulfate.